Single-beam scanning using galvanometers is common in several fields including cellular microscopy. Single-beam scanning-based imaging techniques are relatively slow because the image pixels are acquired sequentially through the raster scanning of a single imaging point. This can be particularly detrimental in the field of high-content screening involving the confocal scanning of thousands of high-resolution cellular images in an automated fashion. In these high-content screening experiments, image acquisition times for a single frame may be of the order of tens of seconds in cases where there are limitations on the signal collected from the sample, as is the case in time-domain fluorescence-lifetime imaging microscopy (FLIM). These screens can involve hundreds of differently treated cellular samples, for example in a 384-well plate, for which multiple images must be taken of different sites within each condition to achieve statistically relevant data. The resulting need for thousands of images translates to multiple hours of imaging. This can for example be an issue in applications where the interaction to be measured is transient, where the sample is not stable, or where a time-course is desired to follow the impacts of the experimental treatment. Similar issues may be encountered in other “photon-starved” optical imaging techniques.
Efforts to improve scan speeds have concentrated on higher speed scanning mechanisms (e.g., rotating mirrors, resonant galvanometers) and multiconfocal (e.g., spinning disk) approaches. However, these approaches generally have drawbacks in terms of reduced spatial resolution and lower signal-to-noise ratios. Furthermore, in situations where the signal is limited by saturation or photobleaching of the analytes with excitation light, further reduction in imaging time is not possible or practical using a faster scan speed. In these cases, the scanning speed is limited not by the speed of the scanning mechanism, but by the rate of photons that can be collected from the illuminated sample region. Conventional methods for improving scanning speed while preserving image quality generally involve one or more of the following: increasing the energy of the collected signal by enlarging the numerical aperture of the collecting optics; improving the throughput of the collection optics; and increasing the quantum efficiency of the detector. The development of techniques for increasing or improving these parameters has been a topic of research for decades, and although improvements have been made, in some cases they are approaching physical limitations.
Another known approach, aimed at increasing the speed of neuron scanning to make time resolved images, is disclosed in Lillis et al. “Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution”, Journal of Neuroscience Methods, volume 172, issue 2, pages 178-184 (2008). In an experiment disclosed in this reference, a pre-image is acquired, and a vector-based scan route is determined to maximize cellular analysis against intercellular spaces. In the intercellular spaces the scanning speed was increased using a maximum acceleration, maximum deceleration approach. This path was then used for repeated imaging of the same sample for rapid updates in a time-course imaging set. This approach is not suitable for high-content screening, however, since it requires a full raster scan image to plan the optimal galvanometric scan pattern for each field of view. A separate high-speed full-field imaging system could be incorporated for this purpose, but has the drawbacks of adding to system cost and complexity, and potentially resulting in photobleaching of some fluorophores prior to imaging.
Challenges therefore remain in the development of scanning imaging techniques that can alleviate at least some of the above-mentioned drawbacks.